Obesity is one of the major health hazards in the United States. The underlying mechanism behind obesity is poorly understood. Leptin, a product of the obese gene, is secreted primarily by fat cells and acts centrally, particularly in the hypothalamus, to reduce food intake and body weight (wt). Since most obese individuals are hyperleptinemic, a state of leptin resistance appears to be the main cause of obesity in humans and rodents. Rodent models of diet-induced obesity (DIO), in which animals become obese and hyperleptinimic with high-fat feeding, appear to be comparable to human obesity. Thus understanding the mechanisms behind the development of DIO in rodents may have direct relevance to the neurobiology of human obesity. Although DIO is associated with central leptin resistance, the mechanisms behind this phenomenon are not clearly understood. Our study suggests that a PI3K-PDE3B-cAMP pathway interacting with the JAK2-STAT3 pathway constitutes a critical component of leptin signaling in the hypothalamus. In a rat model of chronic central leptin infusion in which neuropeptide Y and proopiomelanocortin (POMC) neurons develop leptin resistance, the STAT3 pathway remains elevated but the PI3K-PDE3B-cAMP pathway is compromised in the hypothalamus. Recently, we have shown an impaired PI3K pathway of leptin signaling in the hypothalamus of DIO mice. Thus, a defect in the PI3K-PDE3B-cAMP pathway of leptin signaling could underlie the development of central leptin resistance and DIO. Four specific aims will test this possiblity. Aim 1: To test the hypothesis that the hypothalamic PDE3B-cAMP pathway of leptin signaling is impaired during the development of DIO. Aim 2: To test the hypothesis that brain or ObRb neuron-specific deletion of PDE3B will result in the developemt of obesity. Aim 3: To test the hypothesis that POMC or AgRP neuron-specific deletion of PDE3B will result in the developement of obesity. Aim 4: To test the hypothesis that knockdown of PDE3B in the ARC of adult mice will alter normal energy homeostasis. PDE3B will be deleted using Cre-LoxP technology. PDE3B activity and cAMP levels will be measured by enzyme assay and EIA, respectively. Gene expression will be measured by qPCR and ISH, and protein levels by Western blot. These studies will further our understanding on the mechanisms underlying the development of central leptin resistance and obesity, and therefore will be relevant to the development of therapeutic approaches to obesity and eating disorders. PUBLIC HEALTH RELEVANCE: Leptin produced primarly by fat cells signals nutritional status to key regulatory centers in the hypothalamus and it has emerged as an important signal regulating body weight homeostasis and energy balance. Most obese individuals are hyperleptinimic suggesting a state of leptin resistance. We have thus been engaged in studies aimed at understanding the mechanisms of leptin signaling in the hypothalamus and how they change during the development of obesity. In this regard, we have described a novel leptin-signaling pathway in the hypothalamus involving activation of PI3K and phosphodiesterase 3B (PDE3B) and a decrease in cAMP levels. Our pharmacolgical studies with inhibitors have shown an important role of PDE3B signaling in transducing anorectic and body weight reducing actions of leptin in the hypothalamus. We are now working to establish the physiological role of the PDE3B-cAMP pathway in transducing leptin action in the hypothalamus and during the development of obesity so that this pathway could be targeted for the treatment and or prevention of the obesity and related disorders. Because rodent models of diet-induced obesity (DIO) appear to be more comparable to human obesity, we will examine the role of PDE3B signaling in the hypothalamus in the development of DIO in mice. We will perform in vivo studies with several transgenic mouse models to establish the consequences of brain- or neuron-specifc PDE3B deficiency in the development of DIO in the presence of a low-fat diet or a high-fat diet. Overall, these studies will define the critical role of PDE3B signaling in the hypothalamus in the context of the physiology and pathophysiology of energy homeostasis.